Cloning host organisms

ABSTRACT

Methods and materials for the cloning of DNA, in particular, for the cloning of &#34;unclonable&#34; DNA using genetically engineered host cells. Host cell organisms have been discovered that stabilize and inhibit rearrangement of DNA molecules capable of forming non-standard secondary and tertiary structures. Organisms are engineered to contain at least one mutation which inactivates homologous recombination and at least one mutation in a DNA repair pathway. Examples of such DNA pathways include UV repair pathway, the SOS repair pathway, the mismatch repair pathway, the adaptive response pathway, the heat shock response pathway, the osmotic shock response pathway, the repair pathway of alkylation damage, the repair pathway of uracil incorporation into DNA and pathways involved in maintaining DNA superhelicity. The host organisms of this invention are suitable for cloning DNA capable of forming non-standard and tertiary structures such as found in eukaryotic DNA.

This is a continuation of application Ser. No. 08/051,572, filed Apr.22, 1993, now abandoned, which is a continuation of application Ser. No.07/462,505 filed on Jan. 8, 1990, now abandoned.

BACKGROUND

1. Field of the Invention

This invention relates to certain methods and materials for the cloningof DNA and, in particular, to the cloning of "unclonable" DNA using agenetically engineered host cell. This cloning host organism includesparticular mutations from existing cells which have been found tostabilize tester DNA plasmids, and can be assembled using standardtransducing and plating techniques.

2. Description of Related Art and Introduction to the Invention

The ability to elucidate gene structure and function often depends to agreat extent upon the construction of recombinant DNA librariesaccurately representing the total genome of a subject organism, followedby the cloning of this recombinant DNA. Generally, the total DNA ofcells from the subject organism is isolated and cut into fragments usingspecific restriction enzymes. The fragmented DNA sequences are theninserted into appropriate vectors for subsequent propagation andamplification in a foreign host. Commonly used vectors include plasmids,cosmids or phages. Examples of host cells suitable for propagation offoreign DNA sequences include bacteria (i.e., E. coli, Bacillussubtilin, Pseudomonas aeruginosa, yeast (i.e., Saccharomyces cerevisiae)Drosophilia (i.e., Drosophilia melongaster) mammalian cell lines (CHO,L-cells) human cell lines (i.e., Hela, Baculovirus, plant cell lines.

To date a number of obstacles have limited the establishment or stableinheritance of foreign (non-native) DNA in host cells. One barrier toestablishment of foreign DNA is the presence of restriction endonucleasethat cleave such DNA. See, e.g., Bickle, T. (1982) In: Nucleases (Linn,S. M., and Roberts, R. J., eds.). Cold Spring Harbor Press, Cold SpringHarbor, N.Y.; Raleigh, E. A., and Wilson, G. (1986) Proc. Natl. Acad.Sci. USA 83:9070; and Heitman, J., and Model, P. (1987) J. Bact.169:3243. Genetic inactivation of these restriction enzymes in certainhost cells (restriction minus strains) can result in a more efficientintroduction of foreign DNA via transformation, transduction,electroporation or conjugation.

Despite such genetic manipulation of host cells, however, many DNAfragments, particularly those from eucaryotic organisms, have beendeemed "unclonable." Many of these "unclonable" DNA segments are knownto contain sequences capable of forming non-standard secondary andtertiary structures. See, e.g., Erlich, D. (1989) In: Mobile DNA (Berg,D. E. and Howe, M. M., eds.). ASM Publications, Washington D.C. andSantella, R. M., Grunberger, D., Weinstein, I. B., and Rich, A. (1981)Proc. Natl. Acad. Sci. USA 78:1451. DNA that is capable of formingcruciforms (hydrogen bonded hairpin structures formed from invertedrepeat sequences) and Z-DNA (a left handed zig zag configured DNAresulting from alternating purine-pyrimidine residues), for example, israpidly deleted or rearranged in E. coli. See, e.g., Santella, R. M.,Grunberger, D., Weinstein, I. B., and Rich, A. (1981) Proc. Natl. Acad.Sci. USA 78:1451 and Fuchs, R. P. P., Freund, A. M., and Bichara, M.(1988) In: Methods and Consequences of DNA Damage Processing (Friedberg,E. C. and Hanawalt, P. C., eds.). Alan R. Liss, Inc., N.Y.

Previous attempts to increase the stability of cloning such complex DNAstructures has met with only limited success. See Wyman, A., Wertman,K., Barker, D., Helms, C., and Petri, W. (1986) Gene 49:263-271. Wymanet al. reported that certain mutations in E. coli host cells resulted inan increased fidelity of representation of certain complex eucaryoticDNA sequences. Specifically, this group found that the use of E. colistrains with mutations in homologous recombination pathways (recB, recC,and sbcB, or recD) increased the representation of polymorphic sequencesof genomic DNA. While these authors did not fully characterize thepolymorphic nature of the DNA sequences cloned, the subject sequenceswere thought to contain long segments of inverted repetitions orpalindromes.

Chalker et al. have reported that the mutation of a single E. coli geneinvolved in a secondary pathway of homologous recombinations (sbcC)results in the stable propagation of a long palindromic DNA sequence.Chalker, A., Leach, D., and Lloyd, R. (1988) Gene 71:201-205. In markedcontrast to the study of Wyman et al., however, the Chalker group didnot observe any major effects of mutations in the primary pathway ofhomologous recombination on palindrome stability. Ishiura et al. studiedthe effects of mutant strains of E. Coli on the deletion of genomiceucaryotic DNA during cloning. Ishiura, M., Hazumi, N., Koide, T.,Uchida, T., and Okada, Y. (1989) J. Bact. 171:1068-1074. These authorsreport that only a quadruple combination of host mutations (recB, recC,sbcB and either recJ or recN) prevented the deletion of DNA segmentsduring host propagation. Strains with single mutations in either primaryor secondary pathways of homologous recombination or combined mutationsin recombination and restriction pathways did not prevent deletion.There were no data on the structural configuration of the DNA sequencesemployed.

The data from these studies, taken together, indicate that host cellmutations of recombination pathways have not lead to increased stabilityof certain forms of complex DNA during propagation, and the divergentand often conflicting results noted above indicate that, at present,there is not an identifiable or even ascertainable host strain that issuitable for cloning complex, eucaryotic DNA.

The present inventor has discovered that complex DNA sequences can bestabilized during cloning by utilizing hosts with combined mutations incertain nucleic acid recombination and repair pathways. Described hereinare novel bacterial hosts designed for cloning foreign DNA by combiningDNA repair mutations into host strains deficient in homologousrecombination. Mutations which allow for stabilization of testerplasmids containing complex DNA are identified in existing strains andthen assembled using standard techniques of transduction, screening,selection and propagation. The resulting bacteria can be used tostabilize and clone complex DNA such as contained in eucaryotic genomes.

SUMMARY OF THE INVENTION

This invention provides for the genetic construction of host organismsthat provide a stable environment for cloning foreign DNA moleculescapable of assuming secondary and tertiary structure susceptible ofrearrangement. These hosts are, thus, suitable for cloning DNA moleculescapable of assuming such secondary and tertiary structures. The hostorganisms of this invention provide a stable environment for DNAmolecules containing inverted repeat sequences, capable of formingcruciforms, and DNA molecules containing alternating purine-pyrimidineresidues, capable of forming Z-DNA. The host organisms of the inventionare characterized as being recombination deficient and containinggenetic mutations in DNA repair pathways. In a preferred embodiment,this invention provides for an E. coli host of the genotype recB, recJ,sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr, hsdRMS, endA1, gyrA96,thi, relA1, lac, supE44, {F'proAB, lacI^(Q) ZM15, Tn10}.

The invention further provides for a method of cloning structurallycomplex DNA, such as found in the eucaryotic genome. This methodcomprises isolating DNA from an organism, cutting the DNA intofragments, inserting DNA fragments of that organism into a vector, andintroducing the vector in the insert into the host organism of thisinvention. The method involves the use of a host characterized asrecombination deficient with mutations in DNA repair systems. In apreferred embodiment, the E. coli host used in cloning is of thegenotype recB, recJ, sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr,hsdRMS, endA1, gyrA96, thi, relA1, lac, supE44, {F'proAB, lacI^(Q) ZM15,Tn10}.

This invention further provides for a method of constructing a hostorganism suitable for cloning DNA molecules capable of assumingsecondary and tertiary structures susceptible of rearrangement. Themethod comprises identifying mutations of recombination and DNA repairpathways which stabilize DNA in existing hosts and assembling thesemutations using standard genetic techniques. This invention furtherprovides for construction of a novel strain of E. coli, designatedSURE™. Construction of the SURE™ strain is accomplished by successivelytransducing E. coli ER1451 with bacterial genomes containing mutationsin recombination and DNA repair systems. A preferred method ofconstructing the SURE™ strain is outlined in FIG. 2 and furtherdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 2 is a flow diagram of the steps used to construct a strain of E.coli that is suitable for cloning DNA capable of forming secondary andtertiary structures susceptible of rearrangement. The SURE™ strain wasassembled from ER1451 using sequential steps of P1 transduction (P1)Bochner plating, and conjugal mating. The E. coli strain designation foreach P1 transduction step indicates the source of genetic materialtransduced. The genotypes of these strains are set forth in Table 1.

FIG. 1 is a schematic representation of the recombination pathways of E.coli.

FIG. 3 is a schematic gene map of the inverted repeat tester plasmid,designated pAL28α.

FIG. 4 is a schematic gene map of the Z-DNA tester plasmid system,designated pAL20Z.

DETAILED DESCRIPTION OF THE INVENTION

I. Abbreviations--the following abbreviations are used throughout thespecification and claims.

A. Relevant Genotypes

end--endonuclease

hsd--host specificity restriction system

mcr--restriction system for DNA containing methylated cytosine residues

mrr--restriction system for DNA containing methylated adenine residues

recBC--Exonuclease V. involved in primary homologous recombinationpathway

sbcC--suppressor of recBC

thr--threonine

umuC--uv mutagenesis

uvr--ultraviolet repair (excision repair system)

recJ--exonuclease involved in secondary homologus recombination pathways

lac--an uncharacterized mutation within the lac operon which results incolonies more intensely blue in the presence of the lacZΔM15 mutationand a source of the α-complementation lac segment

B. Other

DNA--deoxyribonucleic acid

E. coli--Escherichia coli bacteria

SOS--genes coding for components of DNA repair systems; inducible by DNAdamage

II. Definitions--Unless otherwise indicated, the terms listed below willbe defined as set forth.

cruciform--hydrogen bonded hairpin structures formed from invertedrepeat sequences of DNA

infection--any process used to introduce DNA into host organisms.Includes transduction, transformation transfection, conjugal mating, andelectroporation.

insertion--any process used to integrate foreign DNA into vectorsinverted repeats--completely or partially identical but oppositelyoriented DNA sequences restriction minus--cell strains lacking genescoding for enzymes which break down foreign DNA

vector--autonomously replicating DNA units into which DNA fragments areinserted for cloning

Z-DNA--a left-handed helical configuration of DNA resulting fromalternating purine-pyrimidine residues

Construction of mutant bacterial strain

DNA molecules, capable of assuming nonstandard secondary and tertiarystructures, as commonly found in eucaryotic genomes, are oftencompletely deleted or rearranged during propagation in cloning hostcells. A major reason for this lack of fidelity in cloning may be theexistence in the host cell of mechanisms related to the replication andrepair of DNA. Examples of such mechanisms include: (1) pathwaysdesigned for homologous or non-homologous recombination; (2) restrictionsystems which cleave foreign DNA sequences; (3) endonucleases whichcatalyze the breakdown of DNA; and (4) systems designed for repair ofDNA. Both procaryotic and eucaryotic organisms have developed anextensive array of systems designed for DNA repair. Examples of suchrepair systems include: uv repair pathway; SOS repair pathway; mismatchrepair; adaptive response; heat shock response; osmotic shock response;repair of alkylation damage; repair of uracil incorporation into DNA;and gene products involved in maintaining DNA superhelicity. The role ofsuch repair systems in cloning of foreign DNA has not yet beeninvestigated. As noted above, prior attempts to increase stabilizationof foreign DNA have focused only on host cell mutations in therecombination and/or restriction pathways.

The inventors have engineered a host organism with mutations in repairas well as recombination pathways. The resulting host cells serve tostabilize various complex forms of DNA during propagation.

The new strain has been designed to be a superior host for optimizingthe construction of cDNA and genomic DNA libraries as well as forincreasing the probability that the individual clones obtained willaccurately reflect the genetic makeup of the subject organism. Theconstruction of host strains with combined mutations was accomplished bycrossing or mating strains with individual mutations in metabolicpathways. Means of constructing new strains by DNA introduction ormutation include, but are not limited to, Hfr mating, generalizedtransduction, specialized transduction, conjugation, transposonmutagenesis, oligonucleotide-directed mutagenesis, and transformationfollowed by genetic recombination. Examples of transducingbacteriophages include, but are not limited to, P1 or lambda. Details ofa preferred method of construction using P1 phage transduction arepresented in Example 1. Screening and identification of mutant strainsincorporating and expressing the transduced foreign DNA can beaccomplished by any number of methods. Examples of such identificationand screening methods include, but are not limited to, screening forphenotypic traits such as antibiotic sensitivity, drug resistance, ordependency on nutrient sources.

The parent strain, used as the basis for construction, can be anyexisting strain with a well-defined genotype that preferably includes atleast one of the specific mutations sought to be present in the finalgenotype. Numerous such strains are available and have been wellcharacterized. See, e.g., Ishiura, M., Hazumi, N., Koide, T., Uchida,T., and Okada, Y. (1989) J. Bact. 171:1068-1074. The parent strain isthen transduced with or mated with DNA from a second strain known tocontain at least one additional desired mutation. Those progeny cellscontaining the desired mutations from both the donor and recipient cellare identified, propagated and used through subsequent transduction ormating steps. Once the organism is identified which contains all of thedesired mutations, such an organism can be additionally manipulated tomake it suitable with a variety of vectors and screening procedures. Theinvention is a propagatable cell containing combined mutations in bothrecombination and DNA repair systems. Preferably, the cloning host cellalso contains mutations in the endonuclease and restriction systems,which mutations increase the utility of host cells for cloning purposes.

In a preferred embodiment, the inventors have engineered and constructeda novel strain of E. coli, with combined mutations in recombination andrepair systems, that stabilizes complex foreign DNA during propagation.E. coli strain ER1451, known to have mutations of certain DNAendonucleases and components of the restriction pathway, wassequentially transduced with bacterial genomes having the mutationssought to be introduced. In each case, the transduced DNA also containeda gene for tetracycline resistance. Following each individualtransduction step, strains containing the combined mutations werescreened on Bochner plates in order to isolate tetracycline sensitivederivatives which would then serve as a recipient for the nexttransduction step. Successive transduction steps can be performed in anyorder. In a preferred embodiment, the ER1451 strain was transducedsequentially with DNA containing mutations in the recombination pathway,ultraviolet repair pathway, SOS repair pathway, the restriction pathway,a tetracycline-resistant episome carrying a blue/white screeningcassette and a lactose sensitivity gene. As a result of these sequentialtransduction and screening procedures, a strain of bacteria was createdthat is recombination deficient, restriction minus, endonucleasedeficient, and with mutations in DNA repair systems. Details of thispreferred construction sequence are given in Example 1. The resultingbacterial host, constructed by this method, is known as SURE, atrademark of Stratagene Cloning Systems.

The SURE™ strain constructed by this method was then tested to determineits effect on the stability of cloned DNA sequences capable of formingsecondary and tertiary structures and susceptible to rearrangementduring propagation. Examples of such DNA sequences include, but are notlimited to inverted repeats and alternating purine-pyrimidine residues.

Initial studies were performed to evaluate the effects of the SURE™strain on the stability of inverted repeat DNA sequences, capable offorming structures known as cruciforms. An inverted repeat plasmidtester system was created by modifying plasmid pBR322. This testerplasmid system, designated pAL28α, used chloramphenicol resistance asthe inverted repeat sequence. A variety of strains of E. coli, existingstrains as well as the SURE™ strain, were infected with a testerinverted repeat plasmid. Inverted repeats were found to be rapidlyrearranged in virtually all existing strains tested. In contrast to theresults with existing strains, however, the SURE™ strain was found toprovide a stabilizing environment for these inverted repeat sequences.The percentage of rearrangement was decreased 20 fold in the SURE™strain when compared to the other strains tested. The SURE™ strain was,therefore, engineered to carry mutations blocking pathways responsiblefor repairing DNA lesions. Preferred specific mutations for blockingthese two key pathways are uvrC and umuC respectively, althoughmutations in other genes of this pathway or mutations in other repairpathways would give the desired effect. This presence of these mutationswas demonstrated to result in a 10 to 20 fold increase in stability ofDNA containing long inverted repeats.

Z-DNA tester plasmids were created as derivatives of plasmid pBR322. ThepBR322 plasmid, containing sequences for tetracycline and penicillinresistance, was modified to include a Z-DNA segment in the promoterregion for chloramphenicol resistance. The newly created tester plasmidsystem was designated pAL20Z. This tester DNA plasmid was infected intoexisting strains of E. coli as well as the newly constructed SURE™strain. The Z-DNA containing segments were quickly deleted in theexisting strains while, in the SURE™ strain, deletion was significantlyand markedly reduced. These data demonstrate that a combination ofmutations in recombination and repair pathways, such as engineered inthe SURE™ strain, provide a stabilizing influence to DNA capable offorming nonstandard secondary and tertiary structures.

In E. coli, homologous recombination is a complex, multicomponentprocess that can proceed by three interdependent pathways (see FIG. 1).These 3 pathways, recBCD, recE and recF, all require the recA geneproduct and a set of accessory proteins. A standard recA host,therefore, is completely deficient in homologous recombination.Recombination in a recB strain (the recBCD pathway is the primarypathway in a wild type E. coli) is reduced to approximately 0.5%. Theresidual activity is due to the presence of the recE and recF alternatepathways which do not involve recB. A recB recJ double mutant, however,is virtually identical to a recA strain in its recombination deficiency;recJ is required for both alternate pathways. To model this,recombination can be blocked in host organisms with a variety ofmutations. In addition to the ones set forth above, examples of suchalternate mutations include recB, recO and recB, recJ, recN.

The inventors discovered that insertion of either the recA or recBmutations into a sbcC, recJ, umuC, uvrC strain, stabilized both Z-DNAand inverted repeats. The recB derivative was found to exhibit superiorstabilization effects. This discovery, that combined mutations inrecombination and DNA repair pathways, stabilize complex DNA duringpropagation is significant.

The cloning host organism of the instant invention is restriction minus,preferably carrying, in E. coli, hsd, mcr and mrr mutations. The absenceof mcr and mrr restriction activity increases the size andrepresentation of libraries constructed with methylated orhemimethylated DNA. The absence of hsd activity increases the size andrepresentation of libraries constructed from sequences containing EcoKrecognition sequences. An end mutation (inactivating a DNA endonuclease)was observed to result in improved quality of plasmid DNA minipreps.

In a preferred embodiment, the host cell of this invention also harborsa tetracycline-resistant F'episome carrying the lacl^(Q) ZM15 cassettemaking it suitable for blue/white screening on plates supplemented withX-gal and IPTG. The uncharacterized mutation present in XL1-Blue cells,which makes both plaques and colonies more intensely blue when a sourceof the α-complementation fragment is introduced, has been added. Thecell is, thus, suitable for plasmid or phage libraries using a varietyof vectors.

The following examples are set forth to assist in understanding theinvention and should not, of course, be construed as specificallylimiting the invention described and claimed herein. Such variation ofthe invention which would be within the purview of those in the art,including the substitution of all equivalents now known or laterdeveloped, are to be considered to fall within the scope of theinvention as hereinafter claimed.

EXAMPLE 1

Construction of an E. coli strain.

A novel strain of E. coli was constructed from existing strainsutilizing standard transduction and plating techniques. In this example,transduction with P1 phage particles was utilized. Bochner plating(Bochner, et al., J. Bacteriol., 143:926, 1980) was then used to screenderivatives for the desired genotype. An outline of the constructionmethod is schematically diagramed in FIG. 2. The genotypes of thevarious E. coli strains used in the construction pathway are set forthin Table 1.

                  TABLE #1                                                        ______________________________________                                        Strain  Genotypes                                                             ______________________________________                                        ER1451  lac-proAB, thi, gyrA96, endA1, hsdR17, relA1,                                 supE44, {F' traD36, proAB, lacI.sup.Q Z M15}                          CES229  recD1009, hsdR, sbcC201, phoR79::Tn10, recA                           JC12166 recB21, recC22, sbcB15, sbcC201, thr-1, leuB6, thi-1,                         lacY1, galk2, ara-14, xyl-5, mtl-1, proA2, his-4,                             argE3, rpsL31, tsx-33, supE44, recJ284::Tn10                          CAG12156                                                                              MG1655 (E. coli K-12 F.sup.-  λ) uvrC279::Tn10                 JC8947  AB1157 (standard E. coli K-12) umuC::Tn5                              AG279   same as JC12166 except recJ.sup.+, recF143, fuc::Tn10                 JH122   GW1000 (standard E. coli K12, mcrB4::Tn10,                                    mrr2::Tn5, hsdR2)                                                     BB4     hsdR514, supE44, supP58, lacY1, galK2, galT22,                                metB1, trpR55, {F'(lacI.sup.Q Z M15::Tn10}                            AG239   thi, gyrA, supE44, lac.sup.- (uncharacterized) Tet.sup.R              AG1     recA, endA1, gyrA96, thi, hsdR17, supE44, relA                        C600    supE44, thi-1, leuB6, lacY1, tonA21, mcrA                             NM621   recD, hsdR, mcrA, mcrB, supE                                          HB101   hsdS20, supE44, ara14, galK2, lacY1, proA2,                                   rpsL20, xyl-5, recA13, mcrB                                           TBI     ara, lac-proAB, rpsL, 80, lacZ M15, hsdR                              DH5α                                                                            recA1, endA1, relA1, thi, hsdR17, supE44, gyrA96                      ______________________________________                                    

ER1451 cells were grown at 37° C. to a density of 5×10⁸ to 1×10⁹cells/ml in media containing tryptone, yeast extract, NaCl and agar (LBmedia) containing 5mM CaCl₂. Cells were harvested by centrifugation andresuspended in 100 mM MgSO₄, 5 mM CaCl₂ (MC media) media at 37° C. for15 minutes. Cells were again harvested by centrifugation and resuspendedin 1/10 the original volume of MC media. 0.1 ml of the resulting cellsuspension was mixed with a P1 phage, containing the genome of E. colistrain CES229, and incubated at 37° C. for 15 minutes. The reaction wasterminated by the addition of 0.1 ml of 0.2M sodium citrate. 0.5 ml ofLB media was added and cultures grown at 37° C. for an additional 60minutes. These incubation conditions were found to be sufficient forexpression of transduced DNA. Cells (5×10⁶) were then plated directlyonto LB plates containing 15 μg/ml tetracycline. Transductants werescreened for the desired phenotype, then plated onto Bochner plates andscreened for tetracycline sensitivity. The culture media for thisscreening procedure contained in final concentration: 10 g/L tryptone, 5g/L yeast extract, 15 g/L agar, 10 g/L NaCl, 10 g/L NaH₂ PO₄, 2 g/Lglucose, 12 mg/L fusaric acid, 0.1 mM ZnCl₂, and 50 mg/L ofchlortetracycline. Cell colonies demonstrating a rapid rate of growthand sensitivity to tetracycline were identified, recovered and utilizedfor subsequent transduction as indicated in FIG. 2. ER1451 cells weresequentially transduced with P1 phages containing the genome of E. colistrains CES229, JC12166, CAG12156, JC8947, AG239, AG279, and JH122. As aresult of these transductions, the following genetic mutations wereassembled into the original ER1451 strain: phoR; sbcC201; recJ; uvrC;umuC; lac*; fuc; recB21; mcrB and mrr. The resulting E. coli strain,AG324, was subjected to F' mating for the purpose of introducing atetracycline resistant F'episome carrying a lacI^(Q) ZM15 cassette foruse in blue/white screening. The final E. coli strain (ATCC depositnumber 55695) constructed has been named SURE™, a trademark ofStratagene Cloning Systems. The genotype of SURE™ is recB, recJ,sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr, Δ(hsdRMS), endA1,gyrA96, thi, relA1, lac*, supE44, {F'proAB, lacI^(Q) ZM15, Tn10}. TheSURE™ strain of E. coli was then used to test for stability of variousstructurally complex DNA sequences.

EXAMPLE 2

Stability of Inverted Repeats

DNA molecules containing an inverted repeat sequence coding forchloramphenicol resistance were created and inserted into modifiedpBR322 plasmid vectors. A gene map of the inverted repeat tester system,designated plasmid pAL28α, is schematically diagramed in FIG. 3.Numerous strains of E. coli, including the SURE™ strain, weretransformed with pAL28α. Transformants were inoculated in mediacontaining only penicillin and grown for up to 100 generations (withserial dilutions). After every 10 or 20 generations, aliquots of cellswere screened on either penicillin containing or chloramphenicolcontaining plates. Cells in which the inverted repeat chloramphenicolresistant sequences were rearranged lost their chloramphenicolresistance. Percentage of cells generating rearrangement of the testerplasmid could, therefore, be readily determined by monitoringchloramphenicol sensitivity. Results from eight of the strains studiedare summarized in Table 2. The genotypes of the strains reported inTable 2 are set forth in Table 1 above.

                  TABLE #2                                                        ______________________________________                                        STABILITY OF INVERTED REPEATS                                                 IN VARIOUS E. COLI STRAINS                                                                Percentage of cells                                                           generating rearranged                                             Strain      plasmid per generation.sup.a                                      ______________________________________                                        AG1         15-20                                                             DH5α  15-20                                                             C600        20-25                                                             NM621       20-25                                                             HB101       15-20                                                             TB1         20-25                                                             DL538       20-25                                                             SURE ™   0.8-1.2                                                           ______________________________________                                         .sup.a Percent rearrangement calculated from the number of cells              maintaining the inverted repeat tester plasmid in its unrearranged            configuration (chloramphenicol resistant) divided by the total number of      plasmidcontaining cells.                                                 

It is evident from the data in Table 1 that only the SURE™ E. colistrain markedly reduced DNA rearrangement. The internal environment ofthe SURE™ strain, therefore, provides a stabilizing influence oninverted repeat DNA sequences. Note that each of the E. coli strainstested contained at least some of the mutations found in the SURE™strain. It is important to note here that even though some strains(i.e,, DH5α) contained mutations in different metabolic pathways(homologous recombination, restriction pathway and endonuclease), suchcombined mutations were not effective in diminishing the rate ofrearrangement. Because the presence of these individual mutations inother strains did not prevent rearrangement of the inverted repeat DNAsequence, it appears as though it is the combination of repair andrecombination mutations in SURE™ that is responsible for increasedstability.

EXAMPLE 3

Stability of Z-DNA

DNA molecules were created which contained alternating purine andpyrimidine residues capable of forming Z-DNA. These Z-DNA containingsequences were inserted into a modified pBR322 plasmid designated pAL20Z(see FIG. 4). A sequence of 26 alternating purine-pyrimidine residues(GC) were inserted into and blocked expression of the promoter regionfor chloramphenicol resistance. Because of the absence of a promoter inthe intact plasmid, host cells containing pAL20Z demonstratechloramphenicol sensitivity. Deletion of the repeating sequences resultsin reconstitution of the promoter and, therefore, expression ofchloramphenicol resistance. A variety of E. coli host strains weretransformed with pAL20Z. Transformants were inoculated into mediacontaining penicillin and grown for up to 200 generations (with serialdilutions). After every 20 generations, 10⁶ cells were plated onto mediacontaining chloramphenicol (50 μg/ml). The percentage of cellsdemonstrating chloramphenicol resistance was taken as a directreflection of the percent of cells rearranging the tester plasmid. Theresults from transformation of seven of these E. coli strains, includingthe SURE™ strain, are shown below in Table 3. The genotypes of these E.coli strains can be found in Table 1.

                  TABLE #3                                                        ______________________________________                                        STABILITY OF Z-DNA IN VARIOUS                                                 E. COLI STRAINS                                                                             Percentage of cells                                                           generating rearranged                                           Strain        plasmids per 25 generations.sup.a                               ______________________________________                                        AG1           15                                                              DH5α    15                                                              C600          10                                                              NM621         20                                                              HB101         15                                                              TB1           10                                                              SURE ™ strain                                                                            <0.1                                                            ______________________________________                                         .sup.a Percent ZDNA rearrangement calculated by determining the percentag     of chloramphenicol resistant cells (indicative of a specific ZDNA             rearrangement) within a population after 25 generations.                 

It is evident from these data that the SURE™ E. coli strainsignificantly and markedly reduced the amount of DNA rearrangement. TheSURE™ strain, therefore, provides a stabilizing environment for Z-DNA.Taken together, the data from Examples 2 and 3 demonstrate that acloning host organism can be engineered and constructed such that itprovides a stabilizing environment for complex DNA molecules capable offorming secondary and tertiary structures. Furthermore, the datademonstrate that this host organism is effective in inhibitingrearrangements of DNA molecules capable of forming a variety of suchsecondary and tertiary structures.

A novel strain of bacteria has been engineered by combining a number ofmutations which significantly reduce the rate of both homologous andnon-homologous recombination within segments of cloned foreign DNA.Non-bacterial DNA, particularly from eucaryotic sources, frequentlycontain sequences capable of forming secondary and tertiary structuressuch as cruciforms (due to presence of inverted repeats) or Z-DNA (foundin alternating purine-pyrimidine stretches). These structures are highlyrecombinogenic in standard bacterial cloning hosts which are incapableof carrying out homologous recombination. Mutations which eliminatecertain repair pathways responsible for excision of or replication pastDNA lesions greatly stabilize these unusual structures. By engineeringmutations which eliminate these pathways into a host strain deficient inhomologous recombination, a novel host that is suitable for cloningforeign DNA has been constructed.

I claim:
 1. A culture of E. coli having ATCC deposit number 55695wherein the E. coli is recB, recJ, sbcC201, phoR, uvrC, umuC::Tn5, mcrA,mcrB, mrr, (MsdRMS), endA1, gyrA96, thi, relAl, lac, supE44, and {F'proAB, lacI^(Q) ZM15, Tn1056 and the E. coli can prevent rearrangements inDNA sequences containing inverted repeats or Z-DNA.